EP0119209A1

EP0119209A1 - Identification of microorganisms

Info

Publication number: EP0119209A1
Application number: EP83902587A
Authority: EP
Inventors: Dyann Fergus Wirth; Diane Mcmahon-Pratt
Original assignee: Harvard College
Current assignee: Harvard College
Priority date: 1982-09-20
Filing date: 1983-07-05
Publication date: 1984-09-26
Also published as: WO1984001174A1

Abstract

On effectue l'identification rapide d'un micro-organisme par l'hybridation d'ADN provenant d'un échantillon contenant des micro-organismes entiers avec un spécimen marqué d'ADN spécifique à l'espèce, ne provoquant pratiquement pas d'hybridation croisée et provenant d'un micro-organisme connu. On diagnostique rapidement des maladies caractérisées par des concentrations localisées de micro-organismes infectieux. L'invention décrit un ensemble permettant d'effectuer ces identifications.Rapid identification of a microorganism is carried out by hybridization of DNA from a sample containing whole microorganisms with a marked specimen of DNA specific to the species, causing practically no hybridization. crossed and from a known microorganism. Diseases characterized by localized concentrations of infectious microorganisms are quickly diagnosed. The invention describes an assembly for performing these identifications.

Description

IDENTIFICATION OF MICROORGANISMS

The invention described herein.was made with Federal Government support and the Government has certain rights in the invention.

This invention relates to identification of a specific microorganism in a specimen or sample containing whole microorganisms and pertains more specifically to the diagnosis of diseases characterized by a localized concentration of infectious organisms in tissue, particularly the skin, or in a biological^' fluid, particularly blood; still more particularly it pertains to the rapid diagnosis of such diseases as leishmaniasis, malaria, trypanosomiasis, babesiasis, and herpesvirus diseases.

In the culturing of microorganisms during the manufacture of such products as pharmaceuticals, e.g. antibiotics, beer, cheese, and the like, it is important to avoid contamination by alien and undesired strains of microorganisms, the presence of which has an adverse effect upon the desired product, and to determine as rapidly as possible the identity of any contaminating strains which may be present. Moreover, there are various classes of diseases each of which is generally characterized by objective symptoms but which may be caused by any one or more of several different closely related infectious organisms. Among such diseases are leishmaniasis (caused by several different species of Leishmania) , malaria (caused by several different species of Plasmodium) , trypanosomiasis (caused by several different species of Trypansoma) , babesiasis (caused by various species of Babesia) , and the diseases caused by various members of the herpesvirus family. The severity and pathogenicity of the disease in each case depends upon the specific identity of the infectious organism. Both the

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Sty iro treatment for the disease and the subsequent medical follow up depend upon rapid, accurate identification of the infecting species. Techniques currently widely used for identification of strains of microorganisms and diagnosis and identification of the infecting species in the case of diseases are time-consuming, involving the isolation and cultivation of the microorganism, e.g. the infectious organism and are often unsuccessful because of serious technical difficulties. Delay in identification is particularly serious in the case of those diseases which are endemic to areas where medical services are not readily available.

It has been proposed by Barker et al., Molec. and Biochem. Parasitology, Vol. 3, 33-46 (1981) and Arnot et a., idem, 47-56 to identify Leishmania organisms by culturing organisms from cutaneous lesions, isolating from the cultures kinetoplast DNA (kDNA) , subjecting it to digestion with a restriction enzyme, then subjecting it to hybridization with labelled kDNA from a known organism. However, specific cross hybridization between three different species was reporte .

Ostrow et al.. Virology, Vol. 108, 21-27 (1981) described extraction and purification of virus from wart tissue, extraction and purification of DNA from the purified virus, and treatment of the purified DNA with restriction enzyme followed by hybridization with labelled DNA from known organisms. Brautigam et al., J. Clin. Microbiology, vol.

12, 226-234 (1980) described, culturing of cells infected with herpes simplex virus, isolation of DNA from the culture, and hybridization with labelled DNA from a known virus. Substantial cross-reactivity between species was reported, requiring quantitative determination of extent of hybridization for identification.

It has now been found that species of microorganisms can be identified in samples of products or in samples of infected tissue or of biological fluid such as blood from infected mammals by immobilizing DNA from said sample on a solid support, subjecting said immobilized DNA to hybridization with a labeled specimen of species-specific non-cross-hybridizing DNA from a known organism, and determining whether hybridization occurs.

Despite the immobilization on the support of all DNA from all of the whole microorganisms present as well as that from the host cells of the sample, when such host cells are present, and despite the lack of any purification step and the omission of any restriction enzyme treatment, no interference occurs either from other non-specific DNA in the microorganisms or from the DNA of the host cells; further, selection for labelling of an appropriate DNA specimen from a gene library for the desired species using conventional hybridization procedures as the criteria for selection, ensures minimal cross-reactivity of DNA between species. In the case of Kinetoplastida such as Leishmania and Trypanosomes, kinetoplast DNA (kDNA) is a species-specific non-cross-hybridizing DNA which can be used effectively as the labelled probe or hybridizing agent. in practicing the invention, a sample of the product or culture suspected to contain an undesired microorganism, or a sample of infected tissue from a skin lesion or a sample of infected blood, both of which contain whole organisms of the infecting species, is simply touched momentarily to any conventional solid support for immobilizing DNA, such as

OMτi ^SNATlθ diazobenzyloxymethyl paper, a nitrocellulose filter, or a solid DNA support sold under the trade name "Gene Screen", treated with aqueous alkali (preferably at least 0.2 M) to expose the DNA from within the organism, and the alkali removed by washing. The sample DNA thus immobilized in a small localized zone of the support and containing all of the DNA of the sample, is then subjected to hybridization with a labelled specimen of species-specific non-cross-hybridizing DNA from an authentic known specimen of an individual species or subspecies of the microorganism. This can be accomplished by contacting the immobilized DNA with the labelled specimen under conventional hybridizing conditions, removing excess non-hybridized labelled specimen, for example by washing, then determining whether any labelled DNA remains hybridized to the immobilized sample. By using a labelled DNA specimen from each of the suspected microorganisms or from each of the known infectious species of organism for each disease, the identity of the causative organism is readily and quickly established by the present invention, since several hybridizations with different labelled DNA specimens can be conducted with separate test samples simultaneously.

When diazobenzyloxymethyl paper is used as the support, immobilization of the DNA occurs simply upon contact of the sample with the support, treatment of the support with aqueous alkali, and washing to remove the alkali. In the case of other supports such as nitrocellulose filters and "Gene Screen" heating to a temperature of at least 50°C for one-half hour or more is preferred as an additional step to ensure bonding or immobilization of the available DNA to the support; lower temperatures can be used but require undesirably long times; higher temperatures up to 90°C or more

OMPI require shorter times, but much higher temperatures tend to destroy the DNA.

Because of the simplicity of the procedure, a kit can readily be supplied for carrying out the test of the present invention in any clinical laboratory or even in the field. Such a kit contains a supply of supports for the samples and a supply of labelled DNA specimens from known organisms. A more complete kit would include a supply of aqueous alkali at least 0.2 M in concentration, and a supply of aqueous buffer. One or more standards may also be included in the kit in the form of a separate supply of DNA from a known source, preferably spotted or immobilized at a known location on the supports, as well as supplies of hybridization buffer and washing buffer solutions, and if desired a supply of photographic film for use with radioactivly labelled DNA specimens.

Any known labels can be employed for the DNA specimens; standard radioactive labelling, as with tritium or P, makes it possible to use conventional scintillation counters for rapid and accurate determination of hybridization or to use photographic film for autoradiographic determination. Sensitivity of the test is such that samples containing no more than 1000 organisms and in some cases as few as 300 organisms can readily be identified by the present invention. The invention can be used for identification of microorganisms in a sample of any culture or product containing the microorganisms in sufficient concentration so that a sample of convenient size for immobilizing upon a support contains at least the minimum detectable number of microorganisms. The invention can also be used for diagnosis or identification of any disease in which the infectious organisms are sufficiently numerous so that a tissue or blood sample of practical size, say 0.1 g, contains at least the minimum detectable number of organisms. Such diseases, as pointed out above, include leishmaniasis, malaria, trypanosomiasis, babesiasis, and herpesvirus diseases. The following examples are intended to illustrate the nature of the invention without acting as limitations upon its scope.

Example Sample Preparation Animals, either Balb/c mice or Golden Syrian hamsters, were infected by subcutaneous infections in the rear hind foot pads of 10 to 10 promastigotes of leishmania. Tissue samples for touch preparations were prepared from animals previously infected with either Leishmania mexicana or Leishmania braziliensis promastigotes. The lesion was excised two to three months after infection, the skin was removed and the tissue cut into 2-3 mm pieces. A single tissue piece was used for each touch preparation on nitrocellulose. The tissue was placed on the nitrocellulose filter for a period of 30 seconds to one minute. The nitrocellulose filter was air-dried and placed in a clean envelope for dry storage until the filter could be processed. The nitrocellulose filter was treated with aqueous 0.5 M NaOH, 1.5 M NaCl solution for 10 minutes at room temperature to expose the DNA, followed by a 10 minute treatment in 3 M Tris-HCl buffer solution, pH 8, at room temperature to remove the alkali. The filter was then air-dried and baked at 80°C for one hour.

Labelled k-DNA Preparation Promastigotes of Leishmania species were cultured in Schneider's Drosophila Medium supplemented with 15 percent heat reactivated fetal calf serum. The stocks of leishmania included several subspecies each of L. tropica, L. mexicana and L. braziliensis. The cells (10 ) in each case were pelleted, washed 2 times in phosphate buffered saline and the kDNA was extracted by resuspending the cells in iysis buffer (0.2 M Nacl, 0.01 M Tris, 0.001 M EDTA, pH 8.0, 10% SDS) , then shearing the chromosomal DNA by passage through a 22 gauge needle; the catenated kDNA was pelleted at 38,000 g for 30 minutes. The pellet was resuspended in a minumum volume of buffer or H₂0, and cesium chloride was added to bring to a final concentration of 1.7 g/ml. The DNA suspension was then centrifuged for 48 hours at 40,000 rpm. Fractions (200 μl) were collected from the bottom of the gradient. The DNA was visualized by mixing 10 1 of each fraction with 10 μ1 of ethidium bromide (1 μg/ μl) and viewed using a source of ultraviolet light. The kDNA fraction was pooled, and dialyzed overnight against 10 mM Tris-Cl pH 8.0, 1 mM EDTA (2 x 4L) .

Purified kDNA specimens (0.2-0.4 μg) were labelled by the method of nick translation in a 50 1 reacvtion mixture containing 50 mM NaCl, 10 mM MgCl₂. 10 mM dithiothreitol, 125 p moles of each deoxynucleotide triphosphate (in most reactions, two radioactive ( 32P) deoxynucleotide triphosphates were used), and 12 units of DNA polymerase I. The reaction mixture was incubated at 15^βC for two hours. The labelled DNA was separated from unincorporated dATP on a chromatographic gel column run in deionized water.

The labelled DNA was denatured by boiling for three minutes and chilled on ice immediately before adding to the hybridization mix described below.

Hybridization

The nitrocellulose filter papers on which the

DNA from the sample lesions was immobilized were each soaked for 2 hours at 40°C in hybridization solution (50% formamide, 5 x SSC, 10 x Denhardts, 100 Vig/ml

OMPI denatured Salmon sperm DNA) to which labelled k-DNA from a selected species of organism was added, then incubated for 12 hours at 42°C, washed in 0.1 x SSC with 0.5% SDS three times for 30 min. at 50°C. Each filter paper was air dried and exposed to XAR-5 film to provide an autoradiograph.

In each case the sample DNA from the lesion hybridized with a labelled specimen of kDNA from the same Leishmania species as the one with which the animal had been infected. No cross hybridization was observed between any subspecies of L. mexicana and L. braziliensis, and cross hybridization between subspecies of L. mexicana and L. tropica did not exceed 1%. Even this minor extent of cross hybridization was eliminated by adding unlabelled kDNA from L. tropica in 40-fold excess to the labelled probe from L. mexicana before hybridization with the immobilized sample. Such cross-hybridization was also eliminated by adding unlabelled kDNA from L. mexicana to the labelled probe from L. tropica before hybridization.

Similar results can be obtained in the case of other diseases caused by organisms of the kinetoplastida such as pathogenic South American trypanosomes as well as in the case of products such as pharmaceuticals, beer, cheese, and the like made by means of microorganisms.

What is claimed is:

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Claims

1. In a method of identifying species of microorganisms in a sample containing a mixture of microorganisms, the steps comprising immobilizing on a solid support the DNA from a sample containing said microorganisms, subjecting said immobilized DNA to hybridization with a labelled specimen of species-specific substantially non-cross-hybridizing DNA from a known microorganism, and observing whether hybridization between said immobilized DNA and said labelled specimen occurs.

2. A method as claimed in claim 1 in which said microorganism is an infectious agent and said sample comprises tissue or biological fluid containing said microorganism.

3. A method as claimed in claim 2 in which said microorganism is a kinetoplastid and said labelled specimen comprises kinetoplast DNA.

4. A method as claimed in claim 3 in which said organism is Leishmania.

5. A method as claimed in any of claims 1, 2, 3 or 4 in which the step of immobilizing comprises bringing said sample into contact with said support, treating said support with aqueous alkali, and removing said aqueous alkali from said support.

6. A kit for identifying species of microorganisms comprising a solid support for immobilizing the DNA from a sample containing said microorganisms, and at least one- labelled specimen of species-specific substantially non-cross-hybridizing DNA from a known microorganism.

7. A kit as claimed in claim 6 in which said microorganism is an infectious agent causing a class of diseases and said sample comprises tissue or biological fluid containing said microorganism.

8. A kit as claimed in claim 7 in which said microorganisms are kinetoplastids and said labelled specimen comprises kinetoplast DNA.

9. A kit as claimed in claims 6, 7 or 8 comprising in addition a supply of aqueous alkali at least 0.2 M and a supply of aqueous buffer.